When the eyes of vertebrate animals have been exposed to light and then the light is extinguished, rod photoreceptors in the neural retina must re-establish a dark-adapted state. A critical step in this process is the intracellular stimulation of the enzyme guanylate cyclase (GC). GC-activating proteins (GCAP1 and GCAP2) modulate GC activation. GCAP activation of GC is regulated by the concentration of intracellular calcium ions [Ca2+]i. Previous studies have localized GCAP1 to rod and cone outer segments, somata and synaptic terminals. In the current study, we used in situ hybridization and immunocytochemistry to localize GCAP2 in human, monkey, and bovine retinas. In human and monkey retinas, the most intense immunolabeling with anti-GCAP2 antibodies was in the cone inner segments, somata and synaptic terminals and, to a lesser degree, in the rod inner segments and inner retinal neurons. In bovine retinas, the most intense immunolabeling was in the rod inner segments, with weaker labeling of cone myoids, somata and synapses. Additional enzymatic assays using a GCAP2-specific antibody confirmed that GCAP1, but not GCAP2, is the major component that stimulates GC in bovine rod outer segments. These results suggest that although GCAP1 is involved in the Ca2+-sensitive regulation of GC in rod and cone outer segments, GCAP2 may have non-phototransduction functions in photoreceptors and inner retinal neurons. Although the role of GCAP1 and GCAP2 mutations in human retinal diseases has yet to be established, mutations in GC are known to cause a severe retinopathy known as Leber's congenital amaurosis. Individuals with loss of function mutations in GCAPs might be expected to exhibit a similar phenotype.